Glia mediated ionic balance in the stem cell niche is required for the proper proliferation of neurogenic tissues and wiring of neural circuits

The cellular niche that surrounds stem cells modulates their self-renewal and proliferative properties. In the nervous system glial cells form part of the neural stem cell niche and express a wide variety of ion channels. While the physiological roles of ion channels in glia have been mainly studied in the mature nervous system, the contribution of glial ion channel function in the niche to nervous system development is poorly understood. To gain insight we explored the function of the Drosophila ClC-a chloride channel since its human ortholog CLCN2 is expressed in glial cells and impairment of its function results in neurodevelopmental disorders. We found ClC-a expressed in the niche in cortex glia. These glial cells are in close association with neurogenic tissues and also wrap neuronal cell bodies as they mature away from the niche. Characterization of loss of function ClC-a mutants showed these animals had smaller brains and widespread axon guidance defects. Here we show that ClC-a is required in cortex glia for the proliferation of neuroepithelial cells and neuroblasts, as well as for neuronal survival. We could also relate the axon guidance defects observed in photoreceptors to impaired proliferation in a neuroblast lineage that generates guidepost glial cells essential for this process. Taken together, our results show that ion channels expressed in glia forming part of the neural stem cell niche can have a significant impact on nervous system development. We propose that ion channels can non-autonomously modulate the number of neural progenitors and the progeny of neuroblast lineages, consequently affecting the correct assembly of neural circuits.

contribution of glial ion channel function in the niche to nervous system development is poorly understood. To gain insight we explored the function of the Drosophila ClC-a chloride channel since its human ortholog CLCN2 is expressed in glial cells and impairment of its function results in neurodevelopmental disorders. We found ClC-a expressed in the niche in cortex glia. These glial cells are in close association with neurogenic tissues and also wrap neuronal cell bodies as they mature away from the niche. Characterization of loss of function ClC-a mutants showed these animals had smaller brains and widespread axon guidance defects. Here we show that ClC-a is required in cortex glia for the proliferation of neuroepithelial cells and neuroblasts, as well as for neuronal survival. We could also relate the axon guidance defects observed in photoreceptors to impaired proliferation in a neuroblast lineage that generates guidepost glial cells essential for this process. Taken together, our results show that ion channels expressed in glia forming part of the neural stem cell niche can have a significant impact on nervous system development. We propose that ion channels can non-autonomously modulate the number of neural progenitors and the progeny of neuroblast lineages, consequently affecting the correct assembly of neural circuits.

INTRODUCTION
Stem cells remarkable proliferative capacity needs to be tightly regulated to ensure the generation of the appropriate amount of cells during development or tissue homeostasis in adulthood. This regulation is controlled in part by stem cell intrinsic programs, but also extrinsic cues provided by the cellular niche that surrounds them. In the nervous system of both vertebrates and invertebrates, glia form part of the niche for neural stem/progenitor cells (Bjornsson et al., 2015;Ruddy and Morshead, 2018). In both systems, the effect of glia on neurogenic tissues has been mainly related to the secretion of factors that regulate the maintenance, proliferation and differentiation of stem and progenitor cells. However, one of the findings that shattered the view of glia as just a passive structural element was the realization that glial cells expressed a large variety of ion channels and neurotransmitter receptors (Barres, 1991;Barres et al., 1990). While physiological roles for several of these ion channels in glia have emerged both in normal and pathological states of the mature nervous system (Black and Waxman, 2013;Nwaobi et al., 2016;Olsen et al., 2015;Pappalardo et al., 2016;Verkhrastsky and Steinhauser, 2000), the contribution of glial ion channel functions specifically in the niche during nervous system development is poorly understood. To address this issue and given the functional parallelisms between vertebrate and Drosophila glia (Chotard and Salecker, 2004;Corty and Freeman, 2013;Freeman and Doherty, 2006), we turned to the fly, where neurogenesis has been extensively studied and the niche is simpler than in vertebrates.
The fly central nervous system contains three structures: the central brain (CB), the ventral nerve cord (VNC) and the optic lobe (OL). The CB and VNC are generated by neural stem cells called neuroblasts that delaminate from the neuroectodem during 4 embryonic development and give rise to larval and adult brain through two rounds of neurogenesis (Doe, 2008). The OL originates from a group of neuroepithelial cells that proliferates and separates into two crescent shaped primordia, the outer and the inner proliferation centers (OPC and IPC), which will produce NBs and precursor cells of the distinct visual processing centers (Apitz and Salecker, 2014). In addition, the OL has been extensively used as a model to address neural circuit assembly (Plazaola-Sasieta et al., 2017). This is mainly because of the modular nature of the fly eye and its stereotyped development, which allow for an easy detection of wiring defects in photoreceptors and other visual system neurons.
The cellular components in the fly niche are the neurogenic cells themselves (neuroepithelia and/or NBs and precursor cells), the newly generated neurons and three types of glia. Among the niche glia, perineural and subperineural glia are components of the blood brain barrier (BBB) that respond to systemic nutritional cues and signal neuroblasts to proliferate (Chell and Brand, 2010;Kanai et al., 2018;Perez-Gomez et al., 2013;Sousa-Nunes et al., 2011;Spéder and Brand, 2014). Cortex glia are large cells that lay below subperineural glia. Both nutritional cues and neuroblast signals induce the remodeling of cortex glia to encase NBs and their immediate progeny in a chamber and older neurons individually (Read, 2018;Spéder and Brand, 2018). This close association protects NBs from oxidative stress and nutritional restriction (Bailey et al., 2015;Cheng et al., 2011), and is essential for neuronal survival (Coutinho-Budd et al., 2017;Dumstrei et al., 2003;Pereanu et al., 2005;Read, 2018;Spéder and Brand, 2018).
In the OL, a distinct subtype of cortex glia that expresses miRNA mir-8 (surface associated cortex glia) is in direct contact with the OPC (Morante et al., 2013). This glia sends signals that regulate the expansion of the neuroepithelium and the timely 5 neuroepithelium to neuroblast transition (Morante et al., 2013). Connectivity is also influenced by glia in the visual system (Chotard and Salecker, 2008;Poeck et al., 2001).
Indeed different types of glial cells control photoreceptor axon pathfinding from the eye to the brain and the targeting of a subset of photoreceptors to the lamina neuropil.
The vertebrate ClC-2 chloride channel has been proposed to be one of the channels involved in K + buffering, a key ionic homeostasis process in which glia are involved (Jentsch and Pusch, 2018). In the mature nervous system, increased neural activity results in an increase of extracellular K + , which can alter neuronal excitability. To lower down the concentration of K + , astrocytes take up the ion and distribute it to distant sites via the astrocytic syncytia. The intake of K + takes place with the concomitant intake of Cland water, producing transient astrocyte swelling (Bellot-Saez et al., 2017). Based on its expression in astrocytic glia, the ClC-2 channel has been proposed to be one of the channels that could participate in this Cluptake (Blanz et al., 2007;Hoegg-Beiler et al., 2014;Sirisi et al., 2017). Mutations in CLCN2 are causative for Leukoencephalopathy with ataxia (LKPAT) (Depienne et al., 2013) and ClC-2 has been related to Megalencephalic leukoencephalopathy with subcortical cysts (MLC) (Hoegg-Beiler et al., 2014;Jeworutzki et al., 2012;Sirisi et al., 2017). Both conditions are characterized by vacuolization of the white matter and edema, most probably a consequence of impaired K + buffering, but in addition patients can also present learning disabilities and mild to moderate mental retardation. The fact that ClC-2 is expressed during development in glial precursors, is required for their differentiation (Hou et al., 2018) and that intellectual impairment can arise from connectivity defects, suggest that this channel could have additional functions during neural development. 6 To test this possibility we focused on ClC-a, the fly homolog of the vertebrate ClC-2 chloride channel (Wang et al., 2017). ClC-a electrophysiological properties have been described and are very similar to its mammalian counterpart (Flores et al., 2009;Jeworutzki et al., 2012). In addition, both ClC-2 and ClC-a are most abundant in epitheliums and the brain. ClC-2 has been shown to play a role in transepithelial transport in enterocytes (Catalán et al., 2004). Similarly, ClC-a is also expressed in the epithelia of the fly digestive system, and it is involved in transepithelial transport in stellate cells of the Malpighian tubules, the fly secretory system (Cabrero et al., 2014;Denholm et al., 2013). In the vertebrate brain, in addition to glia, ClC-2 is expressed in inhibitory neurons where it regulates neuronal excitability (Földy et al., 2010;Ratte and Prescott, 2011;Rinke et al., 2010). We were interested in the observation that ClC-a mRNA is expressed in the developing fly nervous system already at embryonic stages in glia (Kearney et al., 2004;Tomancak et al., 2007Tomancak et al., , 2002 and highly expressed throughout development of the nervous system (Celniker et al., 2009;Rose et al., 2007), which indicated a possible role of the channel in the development of the nervous system.
In this study we have analyzed the expression pattern of Drosophila ClC-a in the brain, characterized the first loss of function mutant alleles of this chloride channel and investigated their effects on the development of the nervous system. We show that ClCa is expressed in several types of glia and uncovered a role for this channel in the niche.
It's expression in cortex glia, which is in close contact with OPC and IPC neuroepithelial cells and NBs, is necessary for the proper mitotic activity of these neurogenic tissues, as well as for the survival of neurons. One of the secondary consequences of reduced neuroblast proliferation was the significantly limited 7 production of guidepost glial cells, which translated into non-autonomous neural circuit assembly phenotypes in photoreceptors. Both neurogenic and connectivity defects could be rescued by glial specific expression of the rat ClC-2 vertebrate channel. We propose that the expression of ion channels in the glial niche can shape the development of the nervous system, both controlling the number of neurons generated as well as and their connectivity.

RESULTS
ClC-a is expressed in two types of glia in the developing brain: cortex glia and ensheathing glia.
To characterize in detail ClC-a expression in the developing brain at different larval stages (L1, L2 and L3) we used reporter lines and antibodies. One of these reporter lines expresses GAL4 under the ClC-a endogenous regulatory sequences (see later in the text) and it recapitulates the ClC-a expression pattern in Malpighian tubule stellate cells observed with an antibody against ClC-a (this study and Cabrero et al., 2014) and a

ClC-a protein trap (ClC-a-GFP) (Supplementary Information and Supplementary
Figure 1A-C). To visualize and follow ClC-a expressing cells along development we combined the ClC-a-GAL4 line with UAS transgenes that outlined the membrane and labeled the nucleus. ClC-a expression was detected in L1 brains on membranes in contact with the developing OPC neuroepithelium ( Figure 1A, B). Colocalization of the nuclear RFP signal with the pan glial nuclear marker Repo indicated that ClC-a was expressed in a subset of glial cells ( Figure 1A, A'). In L2 brains glial membranes started encasing CB neuroblasts ( Figure 1C), and more membrane processes were observed deeper in the brain ( Figure 1D). By late L3 the number of ClC-a + glial nucleus in the CB had greatly increased and their glial membranes confined neuroblasts and their 8 lineages in chambers ( Figure 1E). A slightly deeper section showed ClC-a + glial processes forming a smaller mesh ( Figure 1F) which ensheaths mature neurons. In the OL, the OPC and IPC, which were still producing neuroblasts and progenitors, continued to be surrounded by ClC-a + glial membranes ( Figure 1G). We also noticed a glial process between the developing lamina (i.e. lamina precursor cells or LPC) and the lobula plug (LoP) (Figure 1H), establishing a boundary between these two regions which are innervated by neurons with different origins (i.e. photoreceptors generated in the eye disc and innervating the OL through the LPC area and distal neurons generated from the d-IPC, a region of the IPC) ( Figure 1I). Similar expression patterns were observed with anti-ClC-a antibodies and the ClC-a protein trap, confirming the specificity of the ClC-a-GAL4 driver line in the brain (Supplementary Figure 1D-I).
We next aimed to identify which types of glial cells expressed ClC-a and for that we used cell type specific markers and nuclei position. We concluded that superficial ClCa + nuclei on top of the OPC neuroepithelium correspond to a subtype of cortex glia called surface associated cortex glia (Morante et al., 2013), which lies below perineural and subperineural glia ( Figure 1J). miRNA mir-8 (Karres et al., 2007)), a marker for this subtype of cortex glia (Morante et al., 2013), colocalized with ClC-a protein in cells covering the OPC and the process separating the LPC from the LoP ( Figure 1K).
Additional experiments indicated that ClC-a + nuclei present on the surface of the CB and in cortical areas belong to cortex glia. The membrane and nuclear patterns of ClCa + cells were consistent with the nuclear patterns and the membrane scaffold, also known as trophospongium (Hoyle et al., 1986), observed with the recently described cortex glia driver wrapper (Coutinho-Budd et al., 2017) (compare Figure 1G with Figure 1L). As a matter of fact, there was extensive colocalization between ClC-a + and 9 wrapper + membranes in the CB and OL ( Figure 1M-N), including surface associated cortex glia over the OPC ( Figure 1N, N"). Thus, for simplicity, we will refer to surface associated cortex glia as cortex glia.
In order to assess the presence of glial types other than cortex glia, we used an intersectional strategy whereby only ClC-a + /wrappercells (i.e. non-cortex glia cells) were labeled. This way we identified that ClC-a was also expressed in different subtypes of ensheathing glia such as neuropil-and tract-ensheathing glia. ClC-a was expressed in neuropil ensheathing glia surrounding CB neuropils, including the mushroom body calix ( Figure 1O Taken together these data indicate that ClC-a is expressed already early on in development in cortex glia cells, which are in direct contact with and wrap proliferative tissues such as the neuroepithelia of the OL (OPC, IPC) and neuroblasts in the CB, forming part of the niche. ClC-a is also expressed in different types of ensheathing glia whose processes contribute to the compartmentalization of the brain by demarcating different neuropils and neuronal tracts. 1 0

MiMIC insertions in the ClC-a locus generate strong loss of function alleles.
To explore the role of ClC-a in glia we set out to characterize a set of Minos-mediated integration cassette (Mi(MIC)) insertions into the ClC-a locus ( Figure 3E).
In summary, here we have characterized the first ClC-a mutant alleles and found them to result in strong loss of function of the gene product.

Mutations in ClC-a result in smaller brains with photoreceptor guidance defects.
To address the effect of ClC-a mutations in brain development we started by dissecting adult brains and searching for defects that could have a developmental origin based on ClC-a expression patterns in the larval brain. The observation that ClC-a was expressed in glia over proliferative tissues in the brain (i.e. neuroepithelia and neuroblasts) led us to hypothesize that mutant brains could be smaller than control ones, and to test this idea we measured OLs from control and mutant animals. We indeed observed a reduction in OL size in mutants particularly evident in 05423 ClC-a-GAL4 /Df, the strongest allelic combination, and also present in the 05423 ClC-a-GAL4 /14007 ( Figure 2C) and 14007/Df ( Figure 4H, I).
Given that we detected ClC-a expression in glial processes separating the developing lamina from the LoP and outer chiasm glial cells, we labeled photoreceptors to assess 1 2 their innervation path. The compound eye of the fly is formed by some 800 units called ommatidia. Each ommatidia contains eight photoreceptors, R1-6 that terminate in the lamina forming the lamina plexus, and R7 and R8 that extend to the medulla. As rows of ommatidia form in the eye disc, photoreceptors extend axons and innervate the OL in a sequential manner. This way a retinotopic map forms and each ommatidia in the eye generates a corresponding processing unit in the lamina and the medulla neuropils. This Analysis of ClC-a mutant adult OLs using a pan photoreceptor marker revealed photoreceptor guidance defects. The guidance phenotypes observed could be classified into three levels of severity based on the proportion of R cell axons affected ( Figure   2F). In brains with phenotypes classified as medium, a significant portion of posterior R cell axons bypassed the outer chiasm, projected along the posterior edge of the medulla neuropil turning anteriorly, and extended for variable distances before innervating the medulla neuropil from its proximal face. In many cases this resulted in the lamina neuropil being posteriorly misplaced. Despite the presence of these discreet bundles of misprojected axons that originate posteriorly, the photoreceptor array was maintained and mostly regular. We classified as weak phenotypes instances of few misprojected 1 3 posterior axons. Strong phenotypes were characterized by a severe disruption of the photoreceptor array and a posteriorly located and disorganized lamina. Despite the difficulty of identifying discreet bundles of photoreceptor axons, distal innervation was evident. The penetrance and expressivity of these three degrees of severity was variable depending on the allelic combination analyzed ( Figure 2G). This variability could be explained by the fact that ClC-a mutants were not complete nulls. Detailed analysis of mutant photoreceptors also revealed layer selection defects for R8 and R1-R6 neurons (Supplementary Figure 4A-J).
In order to confirm the requirement of ClC-a in glia we performed cell type specific knock down and rescue experiments. In addition to the ClC-a driver, we also used the general glial driver Repo-GAL4 as an alternative to restrict transgene expression selectively to glia. Using these two drivers the knock down of ClC-a by RNAi phenocopied the photoreceptor phenotypes seen in the mutant ( Figure 2H). Moreover, the expression with both glial drivers of ClC-a and rat CLCN2 cDNA, rescued the photoreceptor phenotypes in whole mutant animals, as well as their developmental delay ( Figure 2I). Although it has been suggested that pupal photoreceptors express ClC-a (Ugarte et al., 2005), we did not observe it with antibodies (data not shown) or reporters used in this study, neither in larval nor pupal or adults stages (Supplementary Figure 2D-G). In addition, the absence of phenotype when knocking down ClC-a in the eye disc or generating a full eye mutant for ClC-a (EGUF-hid approach, data not shown), together with the inability to rescue the guidance phenotype when expressing Figure 4K), confirmed that ClC-a was required in glia for photoreceptor guidance. 1 4 Remarkably, taking advantage of the 05423 ClC-a-GAL4 allele we observed a rescue of both the OL size and photoreceptor guidance phenotypes in 05423 ClC-a-GAL4 /Df, the strongest allelic combination, with both ClC-a and ClCN2 cDNA transgenes ( Figure 2D, I). This result indicated that both the brain size reduction and photoreceptor guidance phenotypes in ClC-a mutants are non-autonomous, dependent on the chloride channel expression in glia and that the fly and rat channel have equivalent functions.

Expression of ClC-a in cortex glia is required for neuroepithelial expansion and
neuroblast divisions, and sufficient to restore brain size.
Aiming to unravel how mutations in ClC-a resulted in smaller brains we first assessed the status of glia in ClC-a mutants. We used the 05423 ClC-a-GAL4 /14007 allelic combination to visualize the membranes and nucleus of glia in the mutant background.
Our analysis showed that the distribution pattern of glial cell bodies on the surface of the brain and deep in the cortex was similar in control and mutant animals. Although the ratio of number of nuclei/hemisphere volume in the mutant was slightly reduced compared to control (Supplementary Figure 5A), importantly, the membrane scaffold looked indistinguishable from the one observed in controls covering the whole hemisphere. Processes of ClC-a mutant cortex glia were in close contact with the OPC and IPC neuroepithelia just like in control animals ( Figure 3A, B, E, F). In addition, both in the OL and the CB cortex glia processes formed the trophospongium. That is individual neuroblasts were enclosed in chambers that enlarged to adapt to their lineage expansion ( Figure 3C, G), and mature neuronal cell bodies were progressively enwrapped by cortex glia processes ( Figure 3D, D', H, H'). From these observations we conclude that mutations in the channel do not result in major morphological changes of the trophospongium formed by cortex glia. 1 5 In turn, these results suggested that ClC-a was instead required for the proper physiology of cortex glia. Cortex glia has been shown to be essential for neurogenesis (Dumstrei et al., 2003), and since cortex glia processes are tightly associated to the OPC (surface associated cortex glia (Morante et al., 2013)) and IPC, we set out to examine whether the small OLs in mutant adult brain ( Figure 2C) were a consequence of defects in these neuroepithelia. Neuroepithelia in the OL start as sheets of cells that divide symmetrically and expand until mid L3 (Ngo et al., 2010). As they do so, they bend along the dorso-ventral axis resulting in a crescent shape structure with the opening pointing posteriorly (Nassif et al., 2003). Already in late L2, while the OPC still grows to expand the pool of prospective neuroblasts, neuroepithelium to neuroblast transition starts taking place. The lateral edge gives rise to LPC and the medial edge to neuroblasts that will produce medulla neurons and glia (Egger et al., 2007(Egger et al., , 2010Ngo et al., 2010;Orihara-Ono et al., 2011;Reddy et al., 2010;Wang et al., 2011aWang et al., , 2011bWeng et al., 2012;Yasugi et al., 2010Yasugi et al., , 2008. Once neuroepithelium divisions stop and the wave of differentiation continues, the OPC will start reducing in size and disappear in early pupal stages when it is all converted into precursors and neuroblasts. A similar process takes place in the IPC where different domains generate neuroblasts or migrating progenitors (Apitz and Salecker, 2015;Hofbauer and Campos-Ortega, 1990) until the neuroepithelium disappears.
We first checked if there were differences in neuroepithelia between control and mutant animals. For this we stained brains with the neuroepithelium marker E-cadherin and manually segmented the tissue to generate a 3D reconstruction of these structures, which gave us information about their morphology ( Figure 4A) and size ( Figure 4B). In 1 6 control animals in the mid L3 stage, the OPC and IPC crescents had their ends close together. In late L3, with the addition of progeny from neuroblasts, the OL was bigger and neuroepithelia crescents wider and thinner. In comparison, in mid L3 mutant animals, while neuroepithelia had the same crescent shape as in controls they were already clearly smaller ( Figure 4B). By late L3, in most cases, the OPC appeared as two separate dorsal and ventral domains with the central part absent. Similarly, part of the IPC was also missing ( Figure 4A).
We next wondered whether the reduction in the size of the neuroepithelial sheets was due to cell death and to test that we stained larval brains with an antibody against the apoptosis marker Dcp-1 (cleaved death caspase protein-1). Although developmental cell death was taking place generally in the brain, we did not observe apoptotic cells neither in control nor mutant neuroepithelial cells in mid or late L3 stages ( Figure 4C). Thus, the absence of cell death in this tissue suggested that defects in proliferation could be the cause for the reduction in size of the OPC and IPC at mid L3, as well as the morphological defects in late L3. The lack of neuroepithelial cells in the OPC central domain could be due differentiation taking place (Supplementary Figure 6) in a neuroepithelium with reduced proliferation hence resulting in a premature disappearance of the tissue. To address proliferation defects we carried out a clonal analysis study. With this technique, once mitotic recombination is induced in a dividing neuroepithelial cell, its progeny is labeled, and thus can be counted. Clones were generated in the L2 stage while the neuroepithelia are dividing and expanding, and their size assessed 48 hours later at mid L3. Neuroepithelia clones generated in the control background (brains where cortex glia expressed the ClC-a channel) had a median size of 1 7 the mutant background (brains where cortex glia did not express ClC-a) were significantly reduced with a median size of 9 and 8 cells for OPC and IPC clones respectively ( Figure 4D). Thus, ClC-a was necessary in cortex glia for neuroepithelial expansion.
Given that ClC-a expressing cortex glia also covers the neuroblasts that originate from the OPC as well as the neuroblasts in the CB we also used clonal analysis to assess neuroblast divisions in ClC-a mutants. The high density and proximity of neuroblasts originating from the OPC makes it difficult to obtain single neuroblasts clones sufficiently apart of each other to be sure that the labeled progeny belongs to a single neuroblast. Thus, we analyzed proliferation in neuroblasts from the CB since they are sufficiently apart from each other. Importantly, both control and mutant animals had the same number of neuroblasts, so CB size reduction in mutants was not due to a decrease in neuroblasts (Supplementary Figure 5B). Using the same clone induction protocol as for neuroepithelial clones, the median size of type I neuroblast clones in the control background was 34 cells, while for clones in the mutant brackground the median size was reduced to 26 cells ( Figure 4E). At this same mid L3 stage we also detected that outside neuroepithelia, which were death free, there was more cell death in mutant than control brains ( Figure 4F). This result suggested that alterations in the physiology of cortex glia in ClC-a mutants affected the trophic role of cortex glia processes that wrap the cell bodies of the more mature neurons of the lineage (Coutinho-Budd et al., 2017;Dumstrei et al., 2003;Pereanu et al., 2005;Read, 2018;Spéder and Brand, 2018). Thus we cannot discard that the reduction in neuroblast clone size could be a combination of both cell death and proliferation defects.

8
Taken together, these data argue that the lack of ClC-a in cortex glia in the niche affects the proliferation of neuroepithelial cells and neuroblasts, as well as for the viability of mature neurons that receive trophic signals from cortex glia. Indeed, consistent with both these observations the size and growth rate of larval hemispheres was reduced in the mutant background ( Figure 4G). Thus, these results are in accordance with both OL ( Figure 4H) and CB ( Figure 4I) of adult ClC-a mutant brains being smaller than those of control flies. Importantly, in addition, expression of ClC-a exclusively in cortex glia was sufficient to rescue the size of both structures in the adult ( Figure 4J, K).
Defects are also observed in the neuroblast lineage that gives rise to ClC-a + ensheathing glia, which are necessary guideposts for photoreceptor axons innervating the medulla.
In an attempt to understand how the non-autonomous photoreceptor guidance phenotype related to ClC-a expression in the OL, we performed a detailed developmental expression analysis in the region where photoreceptor innervation takes place. In control L2 brains, horizontal views showed the OPC and IPC still juxtaposed and ClC-a + cell bodies on the surface of the brain and in the CB ( Figure 1D). In L3 frontal views we observed that a population of glia, which preceded the arrival of photoreceptors axons in the lamina (Dearborn, 2004;Perez and Steller, 1996), progressively positioned amid the expanding region between the OPC and IPC during the early to mid L3 stages ( Figure 5A, B). This population was divided in two sets of nuclei, the ClC-anuclei of satellite glia (Supplementary Figure 8A, B) and a population of ClC-a + nucleus, with lower expression than cortex glia, which we called boundary glia ( Figure 5B). From this seemingly homogenous mid L3 boundary glia population, in late L3 brains two cell types could be distinguished in frontal ( Figure 5C) and 1 9 horizontal views ( Figure 5D, Supplementary Figure 8C): the Xg o and a glial type that had never been described before. We called these cells palisade glia (pag) and they positioned in the same plane as the cortex glia projection and the Xg o forming a continuous glial barrier between the developing lamina and the LoP. We do not know if pag reach to or which type they are in the adult ( Figure 5E). Xg o is considered tract ensheating glia and one glial cell enwraps an average of 15 lamina-medulla fiber tracts (Kremer et al., 2017). Two independent studies have shown that Xg o , as well as Xg i , originate from the type II DL1 neuroblast lineage and migrate to the OL (Ren et al., 2018;Viktorin et al., 2013). We repeated DL1 lineage-tracing experiments and observed that progeny from the DL1 populated the OL following the same temporal pattern as ClC-a + boundary glia (Supplementary Figure 8D-F). Hence, our data supports the idea that boundary glia are DL1 progeny that will differentiate into the newly To study the cause of this strong reduction we first used the earmuff R09D11 genomic enhancer-fragment driven reporter CD4-tdtomato (Han et al., 2011)   . In control brains there are 8 type II neuroblasts, 6 positioned medially (DM1-6) and 2 laterally (DL1/2) closer to the OL ( Figure 5N, O). In mutants, although we observed some brains with instances of DM mispositioning, DL1/2 cluster could be found together and laterally located with respect to the rest of DM neuroblasts ( Figure 5P, Q). However, their position with respect to the OL was sometimes changed.
To assess proliferation defects in the lineage our initial approach was to compare control to mutant DL1 clones. However, even though the clonal analysis protocol used in our study was very similar to those used in other studies where they analyzed type II  Figure 5E with Figure 5K).

2
Both the developmental guidance defects and the adult outcomes of ClC-a mutants are extremely similar to OL specific slit mutants ( Figure 6E-H) and robo3 mutants (Pappu et al., 2011;Tayler et al., 2004). The secreted chemorepellent molecule Slit and the Robo family of receptors (Robo, Robo2, Robo3) have been indeed implicated in preventing the mixing of photoreceptor axons with the axons of distal neurons from the LoP during development, and hence maintaining the compartmentalization of that region of the developing brain (Tayler et al., 2004). While receptors have been shown to be required in neurons, slit reporters suggested that Slit protein in the region could be contributed by Xg o (Pappu et al., 2011;Tayler et al., 2004). The detailed developmental analysis on the assembly of the glial barrier allowed us to unequivocally characterize the slit temporal and cellular expression pattern with respect to photoreceptor innervation. To this end we characterized and used a MiMIC-based protein trap line for Slit ( Supplementary Information, Supplementary Figure 10). Our analysis indicated that Slit was already expressed in boundary glia in mid L3 ( Figure 6I-K) when photoreceptors innervate the brain and their axons come in close proximity with these glial cells. Moreover, removal of one copy of slit enhanced the ClC-a photoreceptor guidance phenotype suggesting a genetic interaction between these two genes ( Figure   6L), and knocking down slit in ClC-a + glia in the barrier recapitulated the photoreceptor guidance defects ( Figure 6M).
Taking into consideration our results and previously published studies (Fan et al., 2005;Pappu et al., 2011;Suzuki et al., 2016;Tayler et al., 2004), we propose that the substantial reduction in boundary glia is most probably a combination of proliferation and migration defects, which results in a significant reduction in Slit protein in the region. As a consequence, photoreceptors that innervate the OL close to the glial 2 3 boundary fasciculate with the axons of neurons in the LoP known to innervate the medulla from its proximal site.
Expression of ClC-a exclusively in cortex glia is sufficient to restore ensheathing glia guidepost cells and rescue photoreceptor guidance defects.
To formally test that ClC-a was sufficient in cortex glia to regulate DL1 proliferation, and assess if ClC-a expression in boundary glia (cell type classified as ensheathing glia) played any role in photoreceptor guidance, we performed a cell type specific rescue experiment. Because there is no reporter described exclusively labeling boundary glia before photoreceptor innervation, we carried out a cortex glia specific rescue. We reasoned that with a cortex glia specific driver we would rescue the generation of boundary glia from DL1 and at the same time avoid ClC-a expression in boundary glia ( Figure 7A). Since in this experiment we could not label boundary glia specifically, we used Repo to mark and count glial nucleus in the region in mid L3, when the first photoreceptors begin to innervate the brain. At this time the glial population is compact and easy to identify, while in late L3 additional ClC-aglia such as epithelial and marginal glia appear in high numbers and complicate the counting. In control animals, mid L3 glia nuclei included ClC-asatellite glia and boundary glia ( Figure 7A, B).
While in mutants the number of glial cells is reduced to half due to the strong reduction of boundary glial cells ( Figure 7A, B), expression of ClC-a exclusively in cortex glia resulted in an almost complete rescue in the number of glial cells present in the barrier region in mid L3 ( Figure 7A, B). More importantly, this boundary glia rescue resulted in the rescue of the photoreceptor guidance phenotype ( Figure 7C). Surprisingly, the autonomous ClC-a expression in boundary glia is not necessary for their viability, for migration from their point of origin in the CB to position themselves in the OL, and for 2 4 Slit secretion, since photoreceptor guidance defects are fully rescued when boundary glia are in their position but do not express ClC-a. Thus, we conclude that the absence of boundary glia and the photoreceptor guidance phenotypes are a secondary consequence of the ClC-a requirement in cortex glia and its function in neuroblast proliferation.

Mutations in ClC-a result in widespread wiring defects.
While we have characterized the origin of the guidance defects seen in photoreceptors, wiring defects are not restricted to this cell type. The position and morphology of neuropils in the visual system of ClC-a animals indicate that the wiring of many other neurons in this system is probably affected as well (compare Figure 5E to K).
Moreover, we have also observed defects in CB structures such as mushroom bodies (MBs). Each hemisphere contains one MB, which is formed by the neurons derived from four special type I neuroblasts that never enter quiescence. These neurons extend dendrites forming the calyx and axons project in a fascicle called the peduncle that splits into two branches called lobes ( Figure 8A). Similar to photoreceptors, mushroom bodies are neural structures highly dependent on glia-neuron interactions. It has been shown that glia wrap the peduncle and the lobes during development (Spindler et al., 2009) and in the adult (Kremer et al., 2017), and that different type II DM neuroblasts contribute glia that associates with the mushroom body (Ren et al., 2018). In control animals, ClC-a + glia surrounded the MB calyx ( Figure 8B) and the peduncle ( Figure   8D). Newly differentiated neurons, which are FasII -, projected their axons through the center of the peduncle generating in a ring-like FasII + pattern labeling the oldest neurons ( Figure 8C). In ClC-a mutant animals, in many cases axons misprojected to the calyx ( Figure 8F) and FasII staining filled in the center of the peduncle suggesting that 2 5 newly generated axons did not project through the center of this structure ( Figure 8G).
In addition, the peduncle was much thinner ( Figure 8G) although it seemed that there still was ClC-a + glia surrounding it. Comparison of control and mutant brains stained with antibody against N-cadherin, which labels neuropils, showed how the calyx, which in controls appeared deep in the brain ( Figure 8E), was more superficial in mutants ( Figure 8I). MB clones ( Figure 8J Figure 8N). In clones with strong phenotypes almost all axons terminated in the calyx and the peduncle was barely visible ( Figure 8M). Interestingly, these defects are very similar to those observed when cortex glia and neuropile glia are eliminated: abnormal mushroom body morphologies including splaying of axons and misguidance, and misshapen superficial calix due to premature fusion of the four MB lineages in the cortical region (Spindler et al., 2009).

Thus, as we have observed for photoreceptor guidance phenotypes, the MB defects in
ClC-a mutants could arise due to a reduced production of glia, ClC-a + or otherwise, associated to mushroom body circuitry. In summary then, since guidance defects in the ClC-a mutant seem to be widespread, we propose that ClC-a requirement for proper circuit assembly is not restricted to the OL but general to the brain.

DISCUSSION
In this study we show that the ClC-a chloride channel function in the glial niche has a non-autonomous but profound effect on two key aspects of neural development: the 2 6 generation of neurons and glia in the appropriate numbers, time and place through its role in regulating the proliferation of neurogenic tissues; and as a consequence, the correct assembly of neural circuits. Importantly, the fact that the fly (ClC-a) and rat (ClC-2) chloride channels rescue brain size and guidance defects, suggests that both can perform the same physiological function. Concomitant defects in neuroblast proliferation and photoreceptor targeting have been observed in other studies (González et al., 1989;Kanai et al., 2018;Zhu et al., 2008) and the Activin signaling pathway has been proposed to be required for the production of the proper number of neurons to enable proper connection of incoming photoreceptors axons to their targets (Zhu et al., 2008). Interestingly, mutations in the proneural gene asense, which is expressed in type I neuroblasts and INPs, has adult targeting phenotypes extremely similar to the ones observed in ClC-a mutants (González et al., 1989). Along the same line, our work links ClC-a photoreceptor guidance phenotypes to INP proliferation defects, and moreover, identifies the INP-derived cellular population required for proper photoreceptor axon guidance.
In addition to leukoencephalopathy, patients with mutations in CLCN2 display pathologies associated to cognitive defects. CLCN2 mutant mice also develop widespread vacuolization that progresses with age, but besides photoreceptor and male germ cell degeneration, they do not display immediately visible behavioral defects (Blanz et al., 2007;Bösl et al., 2001;Edwards et al., 2010). However, the fact that CLCN2 is expressed in astrocytes and oligodendrocytes early in development (Makara et al., 2003), and that it has been detected in Bergman glia (Jeworutzki et al., 2012), which is important for neuronal migration in the formation of cortical structures, suggest that it would be worth exploring the role the channel could be playing in   (Jordt and Jentsch, 1997). Since for each molecule of HCO 3 extruded one of Clis internalized, ClC-2 activation might be required to regulate HCO 3 transport allowing for the presence of extracellular Cland this way creating a Clrecycling pathway for HCO 3 -/Clexchangers (Bösl et al., 2001). Assays in Xenopus oocytes concluded that ClC-a activity is also sensitive to pH (H. G-P and R. E unpublished results). Thus, it could be that the lack of ClC-a in cortex glia would lead to a more acidic extracellular pH, due to deficient Clrecycling for HCO 3 -/Clexchangers. Since changes in extracellular and intracellular pH have been shown to affect the proliferative capacity of both wild type and cancer cells (Carswell and Papoutsakis, 2000;Ciapa and Philippe, 2013;Flinck et al., 2018;Persi et al., 2018;2 9 White et al., 2017), ClC-a function in pH regulation could explain the proliferation defects observed in the mutant.
Regardless of the molecular mechanism that mediates ClC-a effect on proliferation our findings contribute to the notion that glia mediated ionic balance is important for brain development. Our findings are in accordance with recent studies suggesting a link between ion channels and the development of the nervous system, with channels being important both in stem cells (Li, 2011;Liebau et al., 2013) as well as glia (Olsen et al., 2015). A recent example of a channel function in stem cells is the gene SCN3A, which codes for NaV1.3 sodium channel. This channel is expressed mostly during development and highly enriched in the basal/outer radial glia progenitors and migrating newborn neurons (Smith et al., 2018). The appearance of this type of progenitors and defined neuronal migration has been associated to the establishment of gyri in the cortex (Fietz et al., 2010;Hansen et al., 2010;Reillo et al., 2011). Intriguingly, mutations in the SCN3A gene result in structural malformations of gyri in the cortex (Smith et al., 2018). Another example is the glia specific Kir 4.1 channel, which is related to neurodevelopmental disorders with associated cognitive defects. Mutations in KCNJ10, which codes for the glial specific Kir4.1 channel, underlie SeSAME/EAST Syndrome (Seizures, Sensorineural deafness, Ataxia, Mental retardation and Electrolyte imbalance/Epilepsy, Ataxia, Sensorineural deafness and Tubulopathy) (Bockenhauer et al., 2009;Scholl et al., 2009) and have been detected in patients diagnosed with autism spectrum disorder and epilepsy (Sicca et al., 2016(Sicca et al., , 2011. The reduction of Kir4.1 expression in astrocytes significantly contributes to the etiology of Rett Syndrome (Kahanovitch et al., 2018;Lioy et al., 2011), which shares many pathophysiological traits with SeSAME/EAST. Moreover, Kir4.1 protein is detected as early as embryonic day 20 in glial cells in the developing cortex and hippocampus (Moroni et al., 2015) 3 0 suggesting that it could influence neural development in those regions. Thus, mutations in ion channels could affect neurogenesis and have an impact in connectivity resulting in intellectual disabilities. Thus, providing insights into the developmental steps affected by impaired glial-dependent homeostasis we can get closer to understanding the origin of cognitive deficiencies detected in patients with channelopathies or conditions where ion channels in glia are not functional. To label membranes and nucleus we used UAS-mCD8-GFP (BDSC 5137), UAS-mCD8-
To generate and label neurogenic tissue clones in the control and ClC-a mutant backgrounds ( Figure 4D,E and Figure 8J) the following stocks were crossed:  When we performed the cortex glia specific rescue experiments ( Figure 4J,K and Figure   7B,C) there was no cortex glia specific driver published, so we devised an intersectional genetic strategy to generate one using the mir8-GAL4 driver. In addition to surface associated cortex glia over the OPC, mir8-GAL4, labels cortex glia and neurons in the brain, as well as other cells in the animal. To restrict mir-8 expression exclusively to cortex glia we combined the following transgenes: repo-FLP6.2 (Stork et al., 2014), tub>GAL80> (BDSC 38879), and mir8-GAL4. In this combination GAL4 is only expressed in cortex glia since the GAL80 repressor has only been flipped out in this cell 3 2 type but stays in non-glial mir-8 expressing cells (Supplementary Figure 7). For simplicity we refer to this combination as mir-8 cxg driver.

DNA constructs
For UAS-ClC-a and UAS-CLCN2 transgenes we used the Gateway cloning System (Invitrogene) and cloned their respective cDNAs into the Φ C31 integrase compatible pBID-UASC-G plasmid (Addgene plasmid # 35202, a gift from Brian McCabe, (Wang et al., 2012)). For the ClC-a construct we used the isoform C (a gift from P. Cid) since its electrophysiological properties had already been studied in Xenopus oocytes and HEK-293 cells (Flores et al., 2009;Jeworutzki et al., 2012) and this isoform has been described to be expressed in Drosophila´s head and body (Flores et al., 2009). The final constructs were injected into the attp40 (25C6) landing site on the 2 nd chromosome.

Immunohistochemistry
Fly brains were dissected in Schneider medium and fixed in 4% PFA in PBL (75mM lysine, 37mM sodium phosphate buffer pH 7,4) for 25 min. After fixation, the tissue was washed in PBS with 0.5%Triton-X-100 (PBST) and blocked with PBST with 10% normal goat serum. Primary and secondary antibody incubations were in PBST and

Photoreceptor phenotype classification
We classified brains as having a strong, medium or weak photoreceptor phenotype based on the OL that out of the two had the more severe phenotype. If not the same, the two OLs tended to be in consecutive categories (i.e. strong/medium, medium/weak, weak/no phenotype). For experiments that entailed photoreceptor phenotypes we always analyzed at least 17 brains.

Measurements and quantifications
To assess adult OL and CB size we took two confocal images of each brain in the appropriate orientations to measure the antero-posterior and dorso-ventral axis of each structure in their widest part. We multiplied those two measurements to obtain a relative value in arbitrary units (a.u). The number of brains analyzed ranged from 23 to 44 for OL (to have fully independent measurements only one OL per brain was quantified) and 12 to 22 for CB when assessing phenotype ( Figure 2C, Figure 4H, I). In rescue experiments ( Figure 2D, Figure 4J, K), the number of brains analyzed ranged from 12 to 32. 3 4 To assess brain size at different larval stages ( Figure 4G), the diameter of one larval brain hemisphere per animal was measured in the antero-posterior axis. The n ranged from 23 to 37 animals analyzed.
To quantify the number of cortex glia nuclei in late L3 OLs we manually counted ClCa + nuclei (Supplementary Figure 5A). Cortex glia nuclei have an average size of approximately 44.5 um2 (Morante et al., 2013) and are clearly distinguishable from ClC-a + ensheathing glia nuclei by their size and position in the OL. The n for this experiment was 8 brains.
To count the number of neuroblasts in the CB part of the hemisphere (Supplementary Figure 5B) we manually identified them based on their nuclear size, and Dpn and Mira antibody staining. Neuroblasts were distinguishable from mature INPs, which are also Dpn + and Mira + , by a smaller nuclear size and their higher intensity of TOPRO-3 nuclear staining. The n for this experiment was 8 brains. To measure neuroepithelia volume the tissue was stained with anti-E-cadherin antibody and manually segmented using the "SURFACE" tool of Imaris software. This tool provides the volume in µm 3 of the generated surfaces ( Figure 4B). The n for this experiment was between 8 and 9 brains.
To quantify the number of cells in OPC, IPC and CB neuroblast clones ( Figure 4D, E) manual counting on confocal stacks was performed. Cells in the clone were identified as TOPRO+ nuclei surrounded by labeled membrane. We counted as many clones as possible per brain as long as they were identifiable as individual clones. The number of clones analyzed in the OPC was of 52 (control) and 31(mutant), in the IPC was 18 (control and mutant), and the number of type I neuroblast clones was 39 (control) and 22 (mutant). 3 5 To assess cell death in developing OLs ( Figure 4F) we manually counted Dcp-1 + /TOPRO + puncta per brain hemisphere. This value was divided by the hemisphere volume obtained through the manual segmentation of the structure and the use of the "SURFACE" tool of Imaris software. The n for this experiment was 6 brains.
To quantify the subset of boundary glia among glial cells in the chiasm region at different stages we manually counted ClC-a + /Repo + nuclei ( Figure 5M). The n for this experiment was 5 brains.
To quantify mature INPs in the DL1 and DL2 lineages ( Figure 5U) we manually counted Dpn + positive nuclei surrounded by tdtom + membranes of the R9D11-tdtomato marker. To differentiate DL1 from DL2 we used gcm-lacZ, which specifically labels the DL2 lineage (Supplementary Figure 9). The n for this experiment was between 11 and 12 brains.
To quantify the number of total glial cells in the future chiasm region in mid L3 ( Figure   7B) we manually counted Repo + nuclei in confocal stacks. The n for this experiment was between 5 and 8 brains.

Image processing
Fiji or Imaris 8.0 (Bitplane, South Windsor, CT, USA) were used to process confocal data as specified in figure legends. Figures were assembled using Adobe Illustrator.

Statistics
Statistical analysis was carried out using Prism 6 (GraphPad Software). When data did not follow normality or resulted from a previous mathematical computation (i.e. ratio to volume) we used non-parametric tests. For group comparisons we used Kruskal-Wallis followed by planned pairwise comparisons with Mann-Whitney post hoc test to obtain 3 6 p-values ( Figure 2C, D; Figure 4B, H, I; Figure 5M; Figure 7B; Supplementary Figure   8H). For pairwise comparisons we used Mann-Whitney's test ( Figure 4D, E, F, J, K, Figure  To evaluate the statistical significance of enhancements in photoreceptor phenotypes qualitatively categorized (i.e. strong, medium, weak, no phenotype) ( Figure 6L, M), we performed a Chi-squared test of independence between phenotype categories and genotypes to obtain p-values. 3 7 project. This work was supported in part by the Spanish Ministerio de Economía y   (A-I) Analysis of ClC-a expression in the developing brain. Brain illustrations show the orientation of imaging planes for the indicated panels at different larval stages. ClC-a specific GAL4 driver (ClC-a-GAL4) was used to label cellular membranes (green) and nucleus (red) of ClC-a + cells. Glial nuclei were labeled with anti-Repo antibody (blue).